Mechanistic deductions from kinetic isotope effects and pH studies of pyridoxal phosphate dependent carbon-carbon lyases: Erwinia herbicola and Citrobacter freundii tyrosine phenol-lyase

Kiick, Dennis M.; Phillips, Robert S.

doi:10.1021/bi00419a023

Cited by 54 publications

(75 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…3 Fifth, very similar pH profiles for D V and D (V/K) were observed for tyrosine phenol-lyase from Erwinia herbicola (35) and led to the assignment of a pK a ϭ ϳ7.6 -7.8 to the catalytic lysyl residue of tyrosine phenol-lyase that abstracts the ␣-proton of L-tyrosine.…”

Section: Discussionmentioning

confidence: 79%

See 1 more Smart Citation

Catalytic Mechanism of the Tryptophan Synthase α2β2 Complex

Ro¹,

Miles

1999

Journal of Biological Chemistry

View full text Add to dashboard Cite

The mechanism of the tryptophan synthase ␣ 2 ␤ 2 complex from Salmonella typhimurium is explored by determining the effects of pH, of temperature, and of isotopic substitution on the pyridoxal phosphate-dependent reaction of L-serine with indole to form L-tryptophan. The pH dependence of the kinetic parameters indicates that three ionizing groups are involved in substrate binding and catalysis with pK a 1 ‫؍‬ 6.5, pK a 2 ‫؍‬ 7.3, and pK a 3 ‫؍‬ 8.2-9. A significant primary isotope effect (ϳ3.5) on V and V/K is observed at low pH (pH 7), but not at high pH (pH 9), indicating that the base that accepts the ␣-proton (␤Lys-87) is protonated at low pH, slowing the abstraction of the ␣-proton and making this step at least partially rate-limiting. pK a 2 is assigned to ␤Lys-87 on the basis of the kinetic isotope effect results and of the observation that the competitive inhibitors glycine and oxindolyl-L-alanine display single pK i values of 7.3. The residue with this pK a (␤Lys-87) must be unprotonated for binding glycine or oxindolyl-L-alanine, and, by inference, L-serine. Investigations of the temperature dependence of the pK a values support the assignment of pK a 2 to ␤Lys-87 and suggest that the ionizing residue with pK a 1 could be a carboxylate, possibly ␤Asp-305, and that the residue associated with a conformational change at pK a 3 may be ␤Lys-167. The occurrence of a closed to open conformational conversion at high pH is supported by investigations of the effects of pH on reaction specificity and on the equilibrium distribution of enzyme-substrate intermediates.The tryptophan synthase ␣ 2 ␤ 2 complex (EC 4.1.2.20) is a useful system for investigating relationships between protein structure and function (for reviews, see Refs. 1-4). The separate tryptophan synthase ␤ 2 subunit 1 and the ␤ subunit in the ␣ 2 ␤ 2 complex catalyze the pyridoxal phosphate (PLP) 2 -dependent conversion of L-serine and indole to L-tryptophan. A number of intermediates in this reaction have been identified from spectroscopic and kinetic studies (see Scheme I in Discussion). The three-dimensional structure of the tryptophan synthase ␣ 2 ␤ 2 complex from Salmonella typhimurium (5) revealed that the active sites of the ␣ and ␤ subunits are ϳ25 Å apart and are connected by a hydrophobic tunnel. This tunnel serves as a passageway for indole from the active site of the ␣ subunit, where it is produced by cleavage of indole-3-glycerol phosphate, to the active site of the ␤ subunit, where it reacts with L-serine to form L-tryptophan. The activity at each site is modulated by reciprocal communication between the ␣ and ␤ sites. These heterotrophic interactions are proposed to switch the ␣ and ␤ subunits between "open" (catalytically inactive) and "closed" (catalytically active) conformations, to coordinate the activities at the two sites, and to prevent the escape of indole (4). Recent crystallographic results provide direct evidence for ligand-induced conformational changes that result in physical closure of loop structures in the ␣ subunit a...

show abstract

Section: Discussionmentioning

confidence: 79%

“…Similar mechanisms are utilized by other PLP enzymes that catalyze ␤-elimination and ␤-replacement reactions (28,31), including O-acetylserine sulfhydrylase (32,33), tryptophan indole lyase (34), and tyrosine phenol lyase (35). The reactions in Scheme I include the release and transfer of three different protons.…”

Section: Discussionmentioning

confidence: 92%

Catalytic Mechanism of the Tryptophan Synthase α2β2 Complex

Ro¹,

Miles

1999

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…This spectrum is similar to that in solution with the exception that the relative intensity of the 330-nm band is much lower in solution. 3 The polarization ratio (i.e. the ratio of absorbance at each wavelength) is shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The amino acid sequence alignment and crystallographic structures show that these two enzymes are very closely related (1,2). Mechanistic studies (3)(4)(5) demonstrated that TPL and Trpase follow very similar catalytic mechanisms (Scheme 3). Both enzymes can catalyze the elimination reactions in vitro of a wide range of amino acids with suitable leaving groups on the ␤-carbon including S-(o-nitrophenyl)-L-cysteine (6,7), S-alkyl-L-cysteines (8,9), ␤-chloro-L-alanine (9, 10), L-Ser (8), and Oacyl-L-serines (7).…”

mentioning

confidence: 94%

Crystals of Tryptophan Indole-Lyase and Tyrosine Phenol-Lyase Form Stable Quinonoid Complexes

Phillips

Demidkina

Zakomirdina

et al. 2002

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

The binding of substrates and inhibitors to wild-typeTyrosine phenol-lyase (TPL) 1 (EC 4.1.99.2) and tryptophan indole-lyase (Trpase, EC 4.1.99.1) are pyridoxal 5Ј-phosphate (PLP)-dependent enzymes that catalyze ␤-elimination reactions to form phenol or indole and ammonium pyruvate from tyrosine and tryptophan, respectively (Schemes 1 and 2) (1, 2). The amino acid sequence alignment and crystallographic structures show that these two enzymes are very closely related (1, 2). Mechanistic studies (3-5) demonstrated that TPL and Trpase follow very similar catalytic mechanisms (Scheme 3). Both enzymes can catalyze the elimination reactions in vitro of a wide range of amino acids with suitable leaving groups on the (8), and Oacyl-L-serines (7). However, in vivo, the enzymes are extremely specific for their respective physiological substrates. Both TPL and Trpase react with substrates and inhibitors to form equilibrating mixtures of external aldimine and quinonoid complexes (5, 10 -14). We have previously reported the x-ray crystallographic structure of TPL complexed with the substrate analog 3-(4Ј-hydroxyphenyl)propionic acid (15). This complex resembles the Michaelis-Menten complex, because the analog lacks an amino group and is unable to form an external aldimine. We would like to obtain structures of quinonoid intermediates for both enzymes, because quinonoid intermediates are proposed to play a central role in the mechanisms of both enzymes (Scheme 3). In previous studies with Trpase and tryptophan synthase (16, 17), we prepared inhibitors oxindolyl-Lalanine (Scheme 4, I) and dihydro-L-tryptophan, which resemble the indolenine intermediate (Scheme 3), a proposed intermediate in the reactions of both enzymes. Trpase and tryptophan synthase are inhibited by different diastereomers of dihydro-L-tryptophan, suggesting that the indolenine intermediates in the reactions of the two enzymes exhibit opposite chirality (17). Oxindolyl-L-alanine was found previously to inhibit Escherichia coli Trpase with a K i value of ϳ2.5-6 M (3, 16, 18) and to form a prominent absorption band at 502 nm, demonstrating the predominant formation of a stable quinon-

show abstract

“…The generally accepted mechanism of TPL catalysis for tyrosinetype substrates [17,25,26] includes the following principal chemical transformations of the substrate: (a) formation of external aldimine; (b) abstraction of the a-proton in the external aldimine; (c) tautomerization of the aromatic moiety to convert it into a good leaving group; (d) and b-elimination of the leaving group (Scheme 1).…”

Section: Discussionmentioning

confidence: 99%

Interaction of tyrosine phenol-lyase with phosphoroorganic analogues of substrate amino acids

et al. 2000

View full text Add to dashboard Cite

The phosphinic analogues of tyrosine and pyruvate were first demonstrated to be substrates in the reactions of elimination and synthesis catalyzed by tyrosine phenol-lyase. Kinetic parameters of the enzymatic process were determined, and the first enzymic synthesis of an aminophosphinic acid was carried out. Replacement of the planar HOOC-group by the tetrahedral (HO)(O)PH-group in the substrate slightly affected its affinity for the enzyme but substantially diminished the conversion rate. For phosphonic analogues, containing (HO) 2 (O)P group, the affinity to the enzyme was decreased considerably while the conversion was completely prevented. Thus, the structural parameters of the acid group are important not only for the affinity for the enzyme, but also for the formation of the catalytically competent conformation of the active site.

show abstract

Mechanistic deductions from kinetic isotope effects and pH studies of pyridoxal phosphate dependent carbon-carbon lyases: Erwinia herbicola and Citrobacter freundii tyrosine phenol-lyase

Cited by 54 publications

References 17 publications

Catalytic Mechanism of the Tryptophan Synthase α2β2 Complex

Catalytic Mechanism of the Tryptophan Synthase α2β2 Complex

Crystals of Tryptophan Indole-Lyase and Tyrosine Phenol-Lyase Form Stable Quinonoid Complexes

Interaction of tyrosine phenol-lyase with phosphoroorganic analogues of substrate amino acids

Contact Info

Product

Resources

About